PROJECT SUMMARY Testosterone and related analogs have been investigated since the 1960s for male contraception. However, an unfavorable side effect profile, that includes cardiovascular liabilities has prevented commercialization. Therefore, the exploration of non-hormonal targets is considered a novel approach to discover contraceptive agents. Despite this very promising possibility a non-hormonal male contraceptive agent has not entered clinical trial or progressed towards IND-enabling preclinical development. Critical to such an approach is the selection of a validated target. In this regard, cyclin-dependent protein kinase 2 (CDK2) is auspicious because CDK2 knockout mice are healthy but sterile. Our central hypothesis is that we can discover an allosteric CDK2 inhibitor lead and a back-up candidate with a different chemical scaffold in the R61 Phase of this project. Furthermore, we hypothesize that we can develop an allosteric CDK2 inhibitor ready for pre-IND development during the R33 Phase. During the R61 phase we propose to discover an allosteric CDK2 inhibitor series and a back-up series preferably with a different chemical scaffold based on already structurally characterized compounds (x-ray co-crystals) and by additional screening. In an iterative fashion, we will optimize and evaluate analogs of existing and new hits by structure-based drug design. Hits will be optimized by using allosteric site binding assays, CDK2 enzyme inhibition assays, ITC, HSQC NMR, X-ray co-crystal structures, kinase selectivity screens, evaluation of physicochemical properties, in vitro ADMET assays and biomarkers in testis explant mouse model. For the R33 Phase, we plan to develop an allosteric CDK2 inhibitor that is ready for pre-IND development. This lead compound should possess selectivity over other kinases, be orally bioavailable, and exhibit reversible meiotic biomarker expression. A back-up compound preferably from a different chemical series will also be progressed to the in vivo oral proof-of-concept stage. Both objectives will be achieved by continuing compound optimization. Promising compounds will be investigated for complete pharmacokinetic properties, off-target effects, and in vivo proof of concept.